Avicenna Journal of Medical Biotechnology
Volume:10 Issue: 4, Oct-Dec 2018

Tissue engineering and regenerative medicine have typically matured from benchtop ideas to commercially applicable products in the clinic 1. However, despite of typical advances in tissue engineering field, some limitations such as no reproducibility, no control of structure geometry including pore size and pore distribution and no integrity of cell distribution and migration in the construct have impelled the scientists into bioprinting technology. The most advantage of 3D bioprinting sounds to be precise fabrication of 3D deposition with controlled geometric structure and cells distribution 2. Over the past decade, lots of researches in bioprinting of different tissues and organs has been carried out using different bioprinting modalities particularly inkjet based printing for skin tissue engineering and extrusion based printing for 3D depositions like bone, cartilage, heart, liver and heart valve. The key factor in extrusion-based bioprinting is bioink preparation, cell encapsulation in the bioink and bioprinting procedure. Indeed, preparation of bioink with appropriate gelation rate, suitable mechanical strength and elasticity which preserve cell viability and proliferation is the most challenge of bioprinting technology. So far, different strategies such as dual bioink cross-linkers, multi-step polymerization and using of core-shell nozzle have been reported to improve viability, quality and functionality of the printed product 3. However, some issues including creation of constructs supporting in vivo vascularization, scaling up tissue constructs and in situ bioprinting have been remained to resolve. A few bioprinting products have been commercialized especially in orthopedic and skin tissue engineering fields and given the fast development of this industry over the past years; it supposed that the bioprinting products will eventually take a big proportion of the medical market to help patients suffering from a wide range of diseases in the future.

Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes Escherichia coli (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems. BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in OrigamiTM E. coli strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line. No difference was observed between oxidoreductase systems in improving the efficiency of protein refolding. However, comparisons between two refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein. A new method for production of biologically active dimeric form of BMP-2 in E. coli expression system was established in this study.

H9N2 avian influenza viruses have the potential to become the next human pandemic threat and next generation vaccine technologies are needed. Current studies introduce nanoparticles as a proper vaccine delivery vehicle for induction of protective immunity. In this study, the efficacy of chitosan nanoparticle-based H9N2 influenza vaccine with and without hemokinin-1 (HK-1) as a molecular adjuvant to induce protective immunity against the virus was examined. The H9N2 antigen was prepared in MDCK cells and inactivated with formalin. The inactivated antigen alone and in combination with HK-1 was encapsulated into chitosan nanoparticles. Groups of BALB/c mice received chitosan nanoparticle-based H9N2 antigen alone or in combination with HK-1 in a prime/boost platform via eye drop method. To evaluate the efficacy of the adjuvanted-nanovaccine candidate, systemic antibody responses were compared among the groups of animals. Serological analysis indicated that mice receiving the HK-1/H9N2 nanoparticles formulation induced higher antibody titers that were sustained until the end of experiment. However, in the immunized mice, influenza specific antibody titers were comparable to that in the animals which were immunized either with inactivated antigen alone or the H9N2 nanoparticles without HK-1 adjuvant. The data demonstrate the synergy between HK-1 as an adjuvant and chitosan nanoparticles as a delivery antigen/adjuvant carrier in the improvement of influenza immune responses.

Previous studies have suggested a protective role for Polyunsaturated Fatty Acids (PUFA) against cancer, cardiovascular, and other diseases. To provide new insights into the in vivo effects of PUFA on gene expression, the effects of dietary PUFA on DNMT3b and PPARα gene expression and global DNA methylation were investigated in selected rat tissues. Thirty sprague-dawley rats were allotted into 3 dietary groups of ten animals each, received experimental diets containing PUFAs every day by gavages for 12 weeks as follows: control group fed a normal diet and water; n-3 PUFAs group received 300 mg/kg/day n-3 PUFAs supplementation; mixed-PUFAs group received 300 mg/kg/day of a mixture of n-3, -6, -9 PUFAs supplementations. The expressions of DNMT3b and PPARα genes were quantitated using real-time RT-PCR. The genome-wide 5-methylcytosine contents in rat tissues were determined by ELISA method. The average expression of the DNMT3b mRNA was 50% lower in the colon and liver of rats fed the n-3- or mixed-PUFAs supplemented diet than control group (p=0.00). However, PPARα expression was significantly upregulated both in the colon and liver of PUFAs-supplemented rats (p<0.001). No significant difference was observed in the blood, colon, and liver DNA methylation levels between PUFAs-supplemented and control animals. The results indicate that dietary PUFAs could modulate the expressions of PPARα and DNMT3b genes in various rat tissues. The findings of this study provide additional insights into the in vivo mechanism of PUFA-mediated regulation of gene expression and could provide an opportunity to develop personalized diets for related disease control.

According to previous studies, Anethum graveolens L. (dill) aqueous extracts decreased the fertility of female rats. Therefore, the present study aimed to examine the effects of this herb on cultured granulosa cells and immature oocytes. The cells were obtained from 27-29 day immature superovulated mice. The oocytes were cultured in a petri dish consisting of 30 μl drops of MEM-α and granulosa cells in a 24-well plate consisting of DMEM/F12 and different concentrations of 0, 10, 50, 100, 500, 1000, 10000 μg/ml of dill seed aqueous extract (DSAE) in 37oC and 5% CO2. Then, the in vitro maturation of oocytes, including Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), and meiosis ІІ (MІІ) and oocyte bioviability were determined. Granulosa cells were then extracted and their bioviability, apoptosis, chromatin condensation, and lipid synthesis were examined. Estrogen and progesterone concentrations and Alkaline Phosphatase (ALP) activity were measured by RIA and spectrophotometry respectively from the supernatant of granulosa cell culture. The results revealed that concentration of 10000 μg/ml of DSAE were toxic and damaged granulosa cell growth and oocytes maturation. Lower concentrations were the same in the control group and did not have any side effects on cell growth. The number of lipid droplets, estrogen and progesterone concentrations, and ALP activity increased with higher doses of DSAE compared to those in the control culture. Additionally, apoptosis and chromatin condensation increased in higher concentrations of DSAE-(500 and 1000 μg/ml) treated cells. This herb extract decreased the oocytes maturation in dose-dependent manner. It was concluded that DSAE increased granulosa cells activity but damaged oocytes maturation, therefore it might be introduced as infertility agent.

The Human Epidermal growth factor Receptor-3 (HER-3) is a member of ErbB receptor family and has deficient kinase activity. HER-3 should heterodimerize with other members of ErbB receptor family, especially with HER-2, to transduce downstream signaling pathways. HER-3 co-expresses with other ErbB receptors in different cancers and overexpresses while the oncogenic signaling pathways such as Jak/Stat, MAPK, and PI3K/Akt are activated and promoted. Here, the expression level of HER-3 was evaluated in Iranian gastric adenocarcinoma's patients and the effects of HER-3 knocking down was investigated on cell cycle and cell viability of human gastric adenocarcinoma cell line of MKN45. In this study, 38 paraffin-embedded surgical adenocarcinoma specimens and their marginal non-tumor tissue samples were collected. Total RNAs were extracted and cDNAs were synthesized. Finally, the expression level of HER-3 was evaluated by real time PCR approach. Moreover, the human adenocarcinoma cell line of MKN45 was transfected with siRNA against HER-3 and the effects of its down-regulation were evaluated using MTT assay and cell-cycle analysis. The data obtained from this study revealed HER-3 is significantly overexpressed in gastric tumors rather than non-tumor marginal tissues. Also, it was found that the expression level of HER-3 is elevated with tumor depth of invasion. Moreover, HER-3 knocking down promotes cell accumulation in G2/M phase of cell cycle and decreases cell viability in MKN45 cells which suggests a potential role for HER-3 in gastric adenocarcinoma tumorigenesis. Taken together, these results emphasize the importance of HER-3 receptor in diagnosis and prognosis of gastric adenocarcinoma.

Alzheimer's Disease (AD) is the most common form of dementia in the elderly. Due to the facts that biological causes of AD are complex in addition to increasing rates of AD worldwide, a deeper understanding of AD etiology is required for AD treatment and diagnosis. To identify molecular pathological alterations in AD brains, GSE36980 series containing microarray data samples from temporal cortex, frontal cortex and hippocampus were downloaded from Gene Expression Omnibus (GEO) database and valid gene symbols were subjected to building a gene co-expression network by a bioinformatics tool known as differential regulation from differential co-expression (DCGL) software package. Then, a network-driven integrative analysis was performed to find significant genes and underlying biological terms. A total of 17088 unique genes were parsed into three independent differential co-expression networks. As a result, a small number of differentially co-regulated genes mostly in frontal and hippocampus lobs were detected as potential biomarkers related to AD brains. Ultimately differentially co-regulated genes were enriched in biological terms including response to lipid and fatty acid and pathways mainly signaling pathway such as G-protein signaling pathway and glutamate receptor groups II and III. By conducting co-expression analysis, our study identified multiple genes that may play an important role in the pathogenesis of AD. The study aimed to provide a systematic understanding of the potential relationships among these genes and it is hoped that it could aid in AD biomarker discovery.

Alzheimer's Disease (AD) is a neurodegenerative disorder, which is the most common cause of dementia in the elderly. Accumulation of β-amyloid plaques outside neurons is the most important pathological hallmark of AD, which is produced by cleavage of amyloid precursor protein by the Alzheimer's β-secretase (BACE1). Since BACE1 is a key enzyme in the formation of β-amyloid peptides, the purpose of this study was to assess the association between polymorphisms of G/C (rs638405) BACE1 gene with sporadic AD in Khuzestan, Isfahan and Fars provinces in Iran. Genotypes were determined by the PCR–Restriction Fragment Length Polymorphism (PCR–RFLP) technique in two groups including 89 sporadic AD patients and 73 healthy subjects. The findings of the BACE1 G/C (rs638405) polymorphism revealed that there was no significant difference between AD patients and controls in men group; however, there was a weak difference in the frequency of CC genotype between patients and controls in women group (χ2=3.333, df=1, p=0.068). The results of this study suggest that the G/C (rs638405) polymorphism of BACE1 gene might not be related with sporadic AD in Khuzestan, Isfahan and Fars provinces in Iran. However, our results do not support a genetic risk factor of this polymorphism for developing AD in male group of this study.

Orofacial cleft is the most common congenital defect of the maxillofacial region. Its non-syndromic type is multi-factorial, and several genes are involved in its occurrence. This study aimed to assess the interaction effect of Rsal and BamHI polymorphisms of Transforming Growth Factor-alpha (TGFα) gene and Bone Morphogenetic Protein-2 (BMP2) and BMP4 variants on the occurrence of Non-Syndromic Cleft Lip and Palate (NSCLP) in the Iranian population. This case-control study was conducted on 120 children with NSCLP and 215 healthy children. Genotyping of the TGFA/BamHI (rs11466297), TGFA/RsaI (rs3732248), BMP4 (rs17563) and BMP2 (rs235768) was performed by Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) methods. Logistic regression was applied to determine the effective factors and the interaction effect of different variants on the occurrence of NSCLP. Gender of patients had no significant association with the occurrence of NSCLP (p=0.335). Multiple logistic regression showed that the interaction effect of the aforementioned polymorphisms on the occurrence of NSCLP was not statistically significant (p=1.000). Although the individual effect of each of the BMP4, BMP2, RsaI and BamHI variants on the occurrence of NSCLP in the Iranian population has been previously confirmed, their interaction does not play a role in this respect.

Infertility is defined as the inability to achieve pregnancy after 12 months of regular unprotected sexual intercourse. Environmental and genetic factors are involved in male infertility. The polymorphism studies have a crucial role in disease recognition. Paraoxonase (PON) is an oxidant enzyme which is associated with inflammation, oxidative stress and lipid metabolism. The present study aimed to evaluate the relationship between PON1 192 Q/R polymorphism and the susceptibility to idiopathic male infertility. Samples were collected from 220 patients diagnosed with male infertility and 230 controls genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). A significant difference in genotype distributions of PON1 192 Q/R polymorphism was observed between patients and controls (p=0.001). Our findings revealed that individuals with the variant QR had a significant decreased risk of idiopathic male infertility (OR=0.49, 95%CI=0.33–0.73, p=0.0004). Moreover, analyses showed that R allele may have a protective effect on susceptibility of idiopathic male infertility (OR=0.31, 95%CI=0.21-0.47, p=0.0001). The data from this study indicates that the PON1 192 Q/R polymorphism is associated with decreased risk of idiopathic male infertility. However, more studies should be considered with larger number of patients and control subjects to confirm our results.

The basis of genetic fingerprinting and DNA profiling in forensic laboratories is the use of Short Tandem Repeats (STRs) according to local and ethnical genetics characteristics. Forensic parameters and allele frequencies for 15 autosomal STRs in 100 unrelated individuals from Khuzestan province, south Iran were determined. PCR was carried out for amplification of STRs and GeneMapper ID software was used for genotyping and allelic analyzing. The Power of Exclusion (PE) varied between 0.332 (TPOX) and 0.768 (FGA). With exception of the THO1 (0.020), TPOX (0.014) and D18S51 (0.003), other STRs showed no deviation from the Hardy-Weinberg equilibrium (p>0.05). Out of 15 STRs, 12 repeats seemed to be more useful and more powerful tools in identity and paternity determination for our studied population. Variation in our data analysis revealed that effective use of these 15 STR loci in forensic cases needed to be localized by collection and analysis of population data from the general population.

Mutations in the coding region of the Chemokine Receptor 5 (CCR5) genes reduce or eliminate CCR5 expression in immune cells and progression of HCV infection. This study aimed to investigate the role of this mutation in HCV infection in Iranian patients in comparison with healthy individuals. 100 HCV infected patients and 100 healthy individuals were randomly selected. The CCR5Δ32 genotypes were determined using specific primers and PCR method. The agarose gel electrophoresis showed a189-bp fragment from wild type for both alleles of CCR5 gene. The CCR5-Δ32 allele was not found in any HCV infected and healthy subjects. The mutation in CCR5 gene was not detected in any of the two groups; therefore, the role of CCR5 gene expression in immune cells and progression of HCV infection needs to be studied in larger samples in our country.

Since the mass production of recombinant proteins requires the development of stable cell lines which is a time-consuming complex process, the use of transient expression on a large scale can be a comparatively useful alternative. Although various cell lines have been used for the expression of recombinant proteins, only a limited number of cells enjoy a high transfection characteristic and the ability to adapt to serum-free suspension culture easily. In the present study, the S2 cells from Drosophila insect with the ability to grow in suspension and serum-free cultures were used for the expression of factor IX (FIX) using Transient Gene Expression (TGE) technique. Drosophila Schneider (S2) cells were seeded in special roller bottles, and then, the cells were transfected with pMT-hFIX plasmid employing the calcium phosphate co-precipitation method. The stable S2-hFIX cells were also seeded in special roller bottles, separately. After the induction, recombinant FIX was quantified in conditioned media employing an ELISA. Moreover, its functional activity was examined using an aPTT assay. The results showed that the expression of FIX through TGE technology was 1.6 times as high as that obtained through S2-FIX stable cells. Furthermore, the comparison of the FIX expression in S2 cells through TGE techniques with that obtained in previous studies in HEK cells or CHO cells revealed that S2 cells were more efficient in terms of FIX expression. The S2 cells with the capability to grow in suspension and serum-free cultures are a suitable alternative for transient expression for the large scale production of proteins.

To improve urinary tract infection detection, we evaluated the specificity and sensitivity of Loop-mediated isothermal Amplification Method (LAMP) for detection of the Eschericia coli (E. coli) in urine samples, for the first time. Primers were designed to target the malB gene of Escherichia coli. LAMP assay was performed on urine specimens collected from patients with urinary tract infection symptoms. As expected, LAMP was more specific and sensitive than direct microscopic tests. LAMP assay showed the best detection limit of DNA copies with 1.02 copies. LAMP method offers several advantages in terms of sensitivity, rapidness and simplicity for detection of E. coli infection in urine samples. The LAMP method would be highly suitable for the early detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment.

Congenital Fibrosis of the Extra Ocular Muscles1 (CFEOM1) is an autosomal dominant condition, caused by mutation in the KIF21A and TUBB3. It is characterized by congenital non-progressive restrictive ophthalmoplegia and ptosis. Mutational analysis of the known genes in such rare diseases by Sanger sequencing not only prevents wasting the time and expenses but also speeds diagnosis process, genetic counseling, and the possibility of prenatal diagnosis. Here, for the first time, association of pathogenic variant c.2860C>T in KIF21A gene in an Iranian family with positive history of CFEOM1A was reported.

We read with interest the article by Fayez et al entitled "Arylamine N-acetyltransferase 2 Polymorphisms and the Risk of Endometriosis" 1 that was recently published in your journal.
These authors evaluated the association between some polymorphisms of human arylamine N-acetyl-transferase 2 (NAT2) gene (in particular C481T, A803- G, G857A and G590A) and the risk of endometriosis development. Specifically, it was found a significant difference in the genotype distributions and allele frequencies of NAT2 G590A polymorphisms between patients with endometriosis and healthy women; in particular, a protective role for NAT2 590A allele in the progression of endometriosis was reported. On the other hand, the authors observed a higher frequency of rapid acetylator phenotype in patients affected.
We deem that this topic appears extremely interesting, but at the same time controversial: it is known that NAT2 enzyme has a critical role in the initial biotransformation of multiple xenobiotic substances (among all aromatic amines and hydrazines) and that several polymorphisms and alleles of its gene characterize the genome of general population (with different distribution in the world) 2. Thus, it appears reasonable an eventual contribution of some NAT2 polymorphisms in the pathogenesis of endometriosis, in which the role oxidative stress (to which the systemic biotransformation of endogenous or exogenous substances may take part) has been largely demonstrated 3. On contrary, previous studies aiming to demonstrate a causative role of NAT2 polymorphisms and acetylator phenotypes in this benign hormone-dependent disease obtained controversial results 4-6.
Although the authors should be congratulated for these innovative findings, we would like to discuss an important aspect of the study. In material and methods, Fayer et al described that DNA was extracted from peripheral blood samples of 141 Iranian patients with surgical confirmation of endometriosis and underwent analysis by PCR-RFLP method. Anyway, authors might specify the different distribution of types of endometriosis in the enrolled population. In particular, it would be of interest to know if women had peritoneal nodules, ovarian endometriomas or deep infiltrating endometriosis (DIE) nodules, or a combination of these. On the other hand, the authors may specifically report if the risk of acetylator phenotypes for having endometriosis has been higher regardless of such endometriosis types. In fact, we think that the polymorphisms of NAT2 gene and the subsequent translated enzymes may differently contribute in these three different types of endometriosis that are known to have distinguished pathogenesis 7. Regarding this topic, it has been previously described that nodules of DIE have higher oxidative stress and inflammatory environment as well more abundant content of nerve fibers, which may cause a more aggressive clinical behavior 8. For this reason, it is conceivable that an anomalous biotransformation of biological endogenous or exogenous macromolecules may give a higher susceptibility for developing DIE.
Anyway, the results of the study by Fayez et al 1 are innovative and promising. Thus, in the near future, new genetic studies are awaited to better clarify the role of NAT2 in the multiple aberrant pathways that characterize endometriotic stromal cells. Additionally, it would be of interest to better understand if this protein (whenever anomalous) may also represent a suitable therapeutic molecular target. In fact, in the last year, research has been particularly active in finding new medical options to treat young women with endometriosis 9 that need a chronic treatment balancing clinical efficacy with safety 10.